The goal of this research is to understand the machinery, which directs co-translational translocation of nascent secretory and membrane proteins. This process occurs at the ER membrane in eukaryotes and at the plasma membrane in bacteria and archaea. The mammalian translocon is comprised of Sec61, which forms the channel and a suite of membrane proteins that include the TRAP complex, the oligosaccharyl transferase complex (OST) and TRAM. When combined with the ribosome, these membrane proteins form a ribosome-channel super-complex at the ER membrane. This project will provide insights into the structure and function of ribosome-Sec61 and ribosome-SecY complexes which act as protein biogenesis engines. In Aim 1, we will extend our model of the ribosome-channel complex by determining the arrangement and structure of Sec61 in non-translating and translating states. In this work, we will combine novel preparative methods with cryo-electron microscopy and single particle methods, to provide models of these complexes. In Aim 2, we will determine the architecture of a ribosome-channel super-complex that contains Sec61, TRAP, OST and TRAM. We will also continue studies of the TRAP complex, which enhances the translocation of many nascent chains. In Aim 3, structures of non-translating and translating ribosome-SecY complexes will be compared. This will clarify the mechanism of co-translational translocation in bacteria and archaea. Finally, a direct comparison of ribosome-SecY and mammalian ribosome-Sec61 complexes should reveal conserved principles of channel assembly and function during protein translocation. PUBLIC HEALTH RELEVANCE: Protein transport channels that associate with ribosomes mediate essential steps in the biogenesis of many proteins (such as peptide hormones, growth factors, apo-lipoproteins etc.), and thus, play important roles in normal and pathological cells. An understanding of these transport channels will provide a framework for studying cellular defects in the relevant pathways. For example, mis-transport of human PrP (prion protein) has been implicated in neurodegenerative disease.